Method for isomerizing glucose syrups

ABSTRACT

The present invention is directed to a method for enzymatically isomerizing glucose in glucose-containing liquors. The presence of relatively small amounts of water-soluble salts of sulfurous acids during the enzymatic isomerization of glucose in glucose-containing liquors reduces color formation therein and increases the stability of the glucose-isomerizing enzyme.

THE INVENTION

The present invention relates to a process of enzymatically isomerizingglucose in glucose-containing liquors. More particularly, the inventionrelates to a process of enzymatically isomerizing glucose inglucose-containing liquors whereby color development in the liquorsduring isomerization is reduced.

The major use of glucose and of corn syrups containing glucose is infood processing, for example in the baking, beverage, canning andconfectionery industries, to provide sweetness, body or to regulatecrystal growth. However, because glucose inherently lacks a high degreeof sweetness and has a relatively bland flavor, its uses are somewhatlimited. This is overcome, to some extent, by mixing glucose or cornsyrups with sucrose or invert syrups to enhance total sweetness. Thishas not proven entirely satisfactory, however, because of economic andother factors involved. It has been recognized that if during theproduction of corn syrups and other glucose-containing syrups asignificant proportion of the glucose could be converted to fructose,syrups would be provided that are sweet enough to satisfy additionalpurposes.

It has long been known in the art that glucose can be converted tofructose by heating a glucose-containing liquor, such as a corn syrup,in the presence of an alkaline catalyst. Because of the nonselectivityof alkaline catalysts various objectionable byproducts are produced,such as large amounts of colored bodies and acidic materials. To refinethe alkaline isomerized liquor to remove the objectionable byproductsthereof, requires rather complicated and costly refining procedures.Consequently alkaline isomerization, as far as we know, has not beenpracticed commercially, due probably, to the economics involved withrefining the alkaline isomerized liquor and the relatively poor qualityof the resulting product.

Various micro-organisms produce poor enzymes which isomerize glucose inglucose-containing syrups to fructose. These enzymes are referred to inthe art as glucose isomerase. An article appearing in Science, Vol. 125,pp. 648-9 (1957) discloses that an enzyme derived from Pseudomonashydrophila will isomerize glucose to fructose. Also British Pat. No.1,103,394 and Japanese Pat. No. 17,640 (1966) disclose thatmicro-organisms classified as belonging to the Streptomyces genus, suchas Streptomyces flavovirens, Streptomyces achromogenes, Streptomycesechinatus, Streptomyces albus, and Streptomyces phaeochromogenes produceglucose isomerase.

Although glucose-isomerizing enzymes are more selective in convertingglucose to fructose than is an alkaline catalyst, there are still anumber of problems associated with the commercial use of these enzymes.For example, appreciable quantities of colored bodies are producedduring enzymatic isomerization, which make the resulting productsdifficult to refine. Also there is a tendency for the isomerizing enzymeto be inactivated in a shorter period than is desired. The formation ofcolored bodies and the inactivation of the isomerizing enzyme arelargely dependent upon the conditions under which the isomerizationreaction is carried out. If the reaction is performed for relativelylong periods of time and/or at high temperatures, in order to obtainhigh yields of fructose, there will be greater amounts of colored bodiesformed and the enzyme will be inactivated to a greater degree.

The purity of the glucose isomerase preparation also affects theformation of colored bodies in the isomerized liquor. If relativelylarge amounts of extraneous materials are present in the glucoseisomerase preparation, there is a greater tendency for larger amounts ofcolored bodies to be formed.

It is the principle object of the present invention to provide anenzymatic method of isomerizing glucose in glucose-containing liquorswhereby the formation of colored bodies in the liquors duringisomerization is reduced.

This object, and other objects of the present invention which will beapparent from the following description, are attained by providing aglucose-isomerizing enzyme in a glucose-containing liquor and subjectingthe liquor to isomerizing conditions, there being present in theglucose-containing liquor during isomerization a small amount of awater-soluble salt of sulfurous acid sufficient to measurably reduce theformation of colored bodies below that level obtained by carrying outthe enzymatic isomerizaton without the presence of the water-solublesalt of sulfurous acid.

Although the present method does not completely eliminate the formationof colored bodies, hereinafter referred to as color, duringisomerization, the small amount of color which is produced may beremoved by relatively simple refining procedures.

In the process of the present invention, the salts of sulfurous acid maybe provided in the glucose-containing liquors by any convenient method.For instance, sulfite or bisulfite salts, or other substances which willgenerate sulfite or bisulfite ions, e.g., SO₂ or H₂ So₃ solution, may beincorporated directly into the glucose-containing liquor before theisomerizaton process is carried out or may be incorporated into theliquor during isomerization. Also sulfite or bisulfite ions may beprovided in the glucose-containing liquors by passing such liquorsthrough ion exchange resins in the sulfite form. Preferably, however thebisulfite and sulfite salts are provided in the glucose-containingliquors before the isomerization process is initiated since the fullbenefit of the presence of these salts will thereby be obtained.

The preferred micro-organisms used to produce glucose isomerase for usein the present isomerization process are those belonging to theStreptomyces genus. The most preferred micro-organism is Streptomycessp. ATCC 21175. The taxonomical characteristics of this micro-organismare shown below.

TAXONOMICAL CHARACTERISTICS OF STREPTOMYCES SP. ATCC 21175A.Morphololigcal CultureMediumObservation__________________________________________________________________________1.spiral sporophores making Yeast extract-malt extract agar;3 to 6 turns;a few incompleted oatmeal agar; glycerol-asparaginesporophores forminghooks and agar; glycerol-starch-glutomateloops. agar; yeast extractglucose agar; tyrosine agar; glycerine syn- thetic agar; calcium malate-glycerine agar; nutrient potato agar; potato plug2. 10 to 50 spores in"sporohores3. Mycelium nonfragmenting "4. Spores surface spiny when"viewed in an electron micro-scope at magnifications above1,000X; sporesround or slightlyelliptical__________________________________________________________________________

B. color of Colony

The most representative color of the spores and aerial mycelia "enmasse" on the surface of mature colonies is beige brown or mist brown,matching color tab 3 ig on the Tresner-Backus color wheel.

C. reverse Side of Colony

No distinctive pigmentation. Gray or brownish yellow on yeastextract-malt extract agar, oatmeal agar and starch agar.

D. color in Medium

No pigment formed.

E. utilization

L-arabinose, D-fructose, i-inositol, D-mannitol, rhamnose, and D-xyloseare utilized for growth. No growth on sucrose and raffinose.

F. other Physiological Properties

Growth is strictly aerobic, mesophilic. No growth at 50° C. on yeastextract-malt extract agar. Proteolytic activity-positive on Gordon andSmith casein agar. Diastatic activity-positive on inorganic salts-starchagar. Volatile compounds with earthy or moldy odor are produced duringthe active growth of the culture on most media.

Another preferred micro-organism belonging to the Streptomyces genusused to produce glucose isomerase is Streptomyces sp. ATCC 21176.

Since glucose isomerase is primarily produced intracellularly by thesemicro-organisms, a source of glucose isomerase may be provided by simplyharvesting the cells from the growth media. The glucose isomerase may beseparated from the cells of these micro-organisms by techniques known inthe art, i.e., sonic treatment, etc., and used to isomerize glucose in aglucose-containing liquor to fructose or the cellular material may beused directly. When cellular material is used there is the tendency formore color and other objectionable byproducts to be produced because ofthe extraneous materials which are present along with the glucoseisomerase, than when separated and purified glucose isomerase is used.However, the techniques necessary to separate the glucose isomerase aregenerally time consuming and involve added expense. Because of this thepresent process is particularly applicable to suppression of colorformation when an enzymatic isomerization reaction is carried out usingas a source of the glucose isomerase cellular material. Since, generallyduring the enzymatic isomerization there is required as enzymeactivators, salts of magnesium, cobalt, chromium, and/or manganese,these salts of sulfurous acid are preferred. In the case of Streptomycessp. ATCC 21175 the preferred salt is magnesium sulfite.

The preferred pH range for performing the enzymatic isomerizationreaction is from about 6.0 to about 8.5 with a pH range of from about6.5 to about 7.5 being most preferred. The temperature of theglucose-containing liquor during isomerization may vary widely, althoughit is preferred that the glucose-containing liquor be at a temperatureof from about 45° to about 80° C. during the isomerization reaction, andmost preferably be at a temperature of from about 50° to about 65° C.

The amount of bisulfite or sulfite salts provided in theglucose-containing liquor may vary, but under the preferred conditionsof the present invention sufficient amounts of these salts are added toprovide an SO₂ content in the liquor of from about 0.02 to about 0.3percent by weight based on the dry substance content of the liquor, andmost preferably from about 0.03 to about 0.07 percent by weight on thesame weight basis. Although at greater concentrations of SO₂ there willbe a relatively long period during the isomerization reaction when lesscolor is produced, than in the case of an isomerization reaction withoutthe presence of sulfites or bisulfites, after this initial period therate of color formation will increase very rapidly until the colorformed will exceed that formed when the isomerization reaction iscarried out without the presence of sulfites or bisulfites. Thereforewhen these salts are used, the isomerization reaction should beterminated before the color formed reaches a point where the subsequentremoval thereof is difficult.

Although the glucose-isomerizing enzyme is relatively stable at hightemperatures it is subject to thermal denaturation normal to allproteins. The presence of the sulfite salts during the isomerizationreaction, especially at high isomerization temperatures, surprisinglyexerts a protective effect towards the glucose-isomerizing enzyme. Thisprovides the benefit that lesser quantities of the enzyme are needed toachieve the same yield of fructose when sulfites or bisulfites arepresent, or conversely for the same quantity of enzyme, higher yields offructose can be obtained.

In order to more clearly describe the nature of the present inventionspecific examples will hereinafter be described. It should beunderstood, however, that this is done solely by way of example and isintended neither to delineate the scope of the invention nor limit theambit of the appended claims. In the examples and throughout thespecification, percentage refers to percent by weight and is based onthe dry substance weight of the glucose-containing liquor unlessotherwise specified.

The analytical methods referred to in the following examples wereperformed as follows:

The color of the glucose-containing liquor was determinedspectrophotometrically by measuring the absorbance of 450 mμ and 600 mμof an appropriately diluted liquor in a 1 cm. cell versus water as areference. The spectrophotometer was a Beckman DK-2A, manufactured byBeckman Instrument Co. The color was calculated by using the followingformula: ##EQU1##

SO₂ CONCENTRATION IN THE GLUCOSE-CONTAINING LIQUOR

Sulfur dioxide in the liquors was determined as follows: A sample of theliquor in the range of 50-60 g. was weighed accurately into a dish andtransferred quantitatively into an 800 ml. Kjeldahl flask employing 300ml. of distilled water. Ten ml. of concentrated phosphoric acid wasadded followed by 1 g. of sodium bicarbonate. The flask was immediatelyconnected to a standard Kjeldahl distillation apparatus andapproximately 250 ml. distilled into a Erlenmeyer flask containing 25ml. of water and 10-12 ml. of 0.8 percent sodium hydroxide solution.When the distillation was complete, the distillate was acidified withphosphoric acid and 2 ml. of starch paste indicator added. The solutionwas then titrated with 0.0625N iodine solution (1 ml. equivalent to0.002 g. of SO₂) until a blue color persisted for 1 minute. Percent SO₂dry basis was calculated as follows: ##EQU2##

Fructose content of the isomerized liquor was determined by measuringthe change in specific rotation which occurred during isomerization.Specific rotations were measured using a Bendix Corporation NPL Model969 Automatic Polarimeter. The rotations were determined at aconcentration of 5 g./100 ml. in a glass cell thermostated at 25° C.Path of the cell was 20 mm. The specifc rotations were determined at thebeginning of the isomerization reactions after all ingredients in theisomerization reaction mixtures had been combined. To determine changein fructose content the specific rotation of the isomerized liquor attime t was determined. All samples were adjusted to pH 4.0 with dilutehydrochloric acid in order to halt enzyme action before dilution fordetermination of rotations. Change in fructose content was calculated byusing the following formula: ##EQU3##

In the formula the factor -138.9 is the change in specific rotationwhich occurs when glucose is converted completely to fructose.

GLUCOSE ISOMERASE ACTIVITY

The determination of glucose isomerase activity of the enzymepreparation is based on a modification of a method disclosed by Takasakiin Japanese Journal of Agr. Biol. Chem., Vol. 30, No. 12, pp.1,247-1,253, using Technicon AutoAnalyzer equipment. The activity of thestandard enzyme used to calibrate the automated procedure was determinedby the method of Takasaki, with the exception that the activity wasdetermined at pH 7.5 instead of 7.2. Thus the definition of a glucoseisomerase unit (GIU) is that amount of enzyme which under the testconditions (pH 7.5, 70° C., 1 hour, test solution 0.1M in D-glucose,0.005M in magnesium sulfate, and 0.05M in pH 7.5 phosphate buffer) willproduce 1 mg. of D-fructose per hour. Fresh cells and dry cells weresuspended in distilled water and sonicated with a Branson Model S75sonifier for 2-3 minutes in order to destroy the cell structure andrelease the enzyme into the liquid phase. The sonicates were centrifugedand appropriate aliquots of the clear supernate taken and diluted to theproper range (0-20 GIU/ml.) for assay by the automated method.

EXAMPLE I

This example illustrates the enzymatic isomerization of glucose inglucose-containing liquors in the presence and absence of sulfite salts.

Streptomyces sp. ATCC 21175 was grown under aerobic submergedfermentation conditions at a pH of 7 in a presterilized aqueous mediumcontaining 1 percent sorbitol, 0.75 percent dextrose, sufficient corncobhydrolysate to provide 1 percent xylose, 4 percent steep water at 29° C.and 0.024 percent cobaltous ion. The fermentation was carried out at 30°C., an airflow of 1 volume of air per volume of medium per minute and aback pressure of 10 p.s.i. The fermenting broth was mechanically stirredat 200 r.p.m. and after 65 hours 4 percent filter aid was admixed intothe broth and the cellular material harvested from the broth byfiltration with suction. The filter cake was washed with demineralizedwater, broken into small pieces and dried for 5 hours in a forced-airoven at an air temperature of 140° F. The activity of the air-driedfilter cake was 660 GIU/g.

A series of four glucose-containing liquors prepared from hydrolysatesof cornstarch were prepared having the compositions shown in thefollowing table:

                                      TABLE I                                     __________________________________________________________________________                   Sample                                                                        1     2     3     4                                            __________________________________________________________________________    Glucose content (percent                                                       dry basis     55.5  55.5  55.5  67.5                                         CoCl.sub.2 6H.sub.2 O (molarity)                                                             0.001 0.001 0.001 0.001                                        Na.sub.2 SO.sub..sub.3 (percent dry basis)                                                   0.25                                                           MgCl.sub.2 6H.sub.2 O (molarity)                                                             0.005       0.005                                              MgSO.sub.3 H.sub.2 O (percent dry                                              basis)              0.25        0.25                                         Total SO.sub.2 (percent dry                                                    basis)        0.13  0.12  none  0.12                                         Glucose Isomerase (GIu/g.                                                      dry basis)    2.3   2.3   2.3   4.6                                          __________________________________________________________________________

These samples were isomerized at a temperature of 70° C. for 92 hourswith the pH thereof being maintained at 6.5 by the addition of a0.5-percent solution of sodium hydroxide. An atmosphere of nitrogen wasmaintained over the three samples which contained the sulfites. Thecolor and the fructose content of the liquors were determined throughoutthe isomerization. The results of these determinations are shown intable II.

                                      TABLE II                                    __________________________________________________________________________            Sample Number                                                                 1        2        3        4                                          Isomerization                                                                         Percent  Percent  Percent  Percent                                    time (hours)                                                                          fructose                                                                           Color                                                                             fructose                                                                           Color                                                                             fructose                                                                           Color                                                                             fructose                                                                           Color                                 __________________________________________________________________________    0       0    6   0    6   0    6   0    7                                     8       2.9  9   5.4  9   3.4  11  9    9                                     20      9.4  10  12.1 11  8.3  36  18   10                                    26      9.8  7   12.7 8   7.4  53  20.2 6                                     44      16.7 10  19.1 14  13.2 96  26.8 17                                    68      21.0 17  22.1 71  14.6 251 30.5 211                                   92      25.0 98  25.3 409 17.5 434 32.5 707                                   __________________________________________________________________________

As seen from table II, as the amount of fructose increased the color ofthe isomerized liquor also increased. In each of the isomerizationreactions carried out in the presence of sulfites less color was formedthan in the liquor which contained no sulfites on an equal fructoseformed basis. Also it is seen that more fructose formed in the samplescontaining the sulfite salts indicating that the sulfites reduced thedegree of inactiviation of the enzyme during the isomerization reaction.

EXAMPLE II

This example illustrates the enzymatic isomerization of glucose inglucose-containing liquors using various amounts of glucose isomerase inthe presence of various amounts of sulfites.

Two series of four glucose-containing liquor samples (mother liquor fromprimary dextrose crystallization, 90DE) were prepared containing 0.005Mmagnesium chloride and 0.001M cobalt chloride. Series A contained 53.4percent dry substance, and Series B contained 56.7 percent drysubstance. To the samples, were added various quantities of theair-dried filter cake of example I and sulfite salts. The isomerizationswere carried out at 70° C. for various times under an atmosphere ofnitrogen. The color and fructose formed during the isomerizationreactions were determined and are shown below in table III.

                                      TABLE III                                   __________________________________________________________________________                         Isomeri-                                                                      zation                                                   Sam-                                                                              Percent                                                                            Percent                                                                            Total  time,                                                                              Percent                                             ple Na.sub.2 SO.sub.3                                                                  NaHSO.sub.3                                                                        SO.sub.2                                                                             hours                                                                              fructose                                                                           Color                                          __________________________________________________________________________    Series A (4.2 GIU/g. of dry substance)                                                             0    0                                                                        17   12.9 11                                                                  29   19.3 35                                             1   0.05      0.025  41   24.8 70                                                                  66   31.9 157                                                                 90   36.8 297                                                                 114  35.8 410                                                                 0    0                                                                        17   14.9 3                                                                   29   22.6 8                                              2   0.05 0.05 0.054  41   26.9 84                                                                  66   33.0 310                                                                 90   36.4 477                                                                 114  36.9 647                                                                 0    0                                                                        17   14.4 4                                                                   29   20.8 7                                              3   0.075                                                                              0.075                                                                              0.081  41   26.4 17                                                                  66   31.8 291                                                                 90   35.8 511                                                                 114  35.8 645                                                                 0    0                                                                        17   14.0 4                                                                   29   20.4 3                                              4   0.10 0.10 0.108  41   26.2 6                                                                   66   32.7 130                                                                 90   36.3 470                                                                 114  37.4 640                                            __________________________________________________________________________    Series B (8.5 GIU/g. of dry substance)                                        __________________________________________________________________________                         0    0    6                                                                   14   21.3 15                                             1   0.05      0.025  25.5 30.6 54                                                                  38   35.5 128                                                                 62   39.2 264                                                                 92   40.5 550                                                                 0    0    5                                                                   14   20.0 9                                              2   0.05 0.05 0.054  25.5 29.5 12                                                                  38   34.7 58                                                                  62   39.5 323                                                                 92   40.5 684                                                                 0    0    4                                                                   14   21.5 8                                              3   0.075                                                                              0.075                                                                              0.081  25.5 30.7 7                                                                   38   35.8 24                                                                  62   39.7 296                                                                 92   41.0 745                                                                 0    0    4                                                                   14   20.5 7                                              4   0.10 0.10 0.108  25.5 29.1 7                                                                   38   35.4 14                                                                  62   39.3 147                                                                 92   41.0 606                                            __________________________________________________________________________

From table III, it is apparent that generally at comparable fructoselevels increasing sulfite content resulted in less color being produced.Also, the user of higher levels of glucose isomerase results in lowercolors at comparable fructose and sulfite levels.

EXAMPLE III

This example illustrates the use of ion exchange resins to providesulfite ions in a glucose-containing liquor and the enzymaticisomerization of the glucose-containing liquor.

A glucose-containing liquor (mother liquor from primary dextrosecrystallization, 90DE) containing 60 g. dry substance per 100 ml. andhaving a color of 8 was heated to 70° C. and sufficient magnesiumchloride and cobalt chloride added to provide a molar concentraiontherein of 0.005 and 0.001, respectively. One-tenth of 1 percent sodiumbisulfite was added and the pH of the liquor was adjusted to 6.5 withdilute sodium hydroxide. A sufficient amount of dried filter cake ofStreptomyces sp. ATCC 21175 prepared according to example I was added toprovide 9.0 GIU/g. dry substance. The liquor was isomerized for 24 hoursat a temperature of 70° C., and the pH during the isomerization wasmaintained at 6.5 by the addition of a dilute sodium hydroxide solution.The fructose content and the color were determined after 22 hours andwere 33.8 percent and 18, respectively. After 24 hours the isomerizedliquor was filtered and divided into four 400 ml. portions each of whichcontained 233 g. dry substance. Each portion was passed separatelythrough ion exchange columns containing various amounts of Dowex 11resin (manufactured by Dow Chemical Co.) in the sulfite form. After ionexchange treatment, the pH of the portions was adjusted to 6.5 with adilute solution of sodium hydroxide and sufficient cobalt chloride addedto give a molar concentration of 0.005. The temperature of the portionswas maintained at 70° C. and at a pH of 6.5 by adding during theisomerization a dilute sodium hydroxide solution. The color and thefructose content during the isomerization reaction were determined andare shown in table IV.

                                      TABLE IV                                    __________________________________________________________________________                                   Cubic feet of                                                                 pound of dry                                                                  resin per                                                                     pound of dry                                                            Isomeriza-                                                                          substance                                                               tion time,                                                                          processed                                                                             Percent                                Sample                                                                            Sample description   hours (×10.sup.4)                                                                     fructose                                                                           Color                             __________________________________________________________________________        Initial Isomerization                                                                              6                  8                                                          22            33.8 18                                                         24    25.0    35.8 7                                 1   After treatment w/ion exchange resin                                                               42    25.0    39.8 10                                                         66    25.0    41.6 31                                                         72    25.0    42.8 63                                                         24    12.5    35.8 9                                 2   After treatment w/ion exchange resin                                                               42    12.5    40.2 15                                                         66    12.5    42.0 72                                                         72    12.5    42.9 173                                                        24    8.3     36.0 10                                3   After treatment w/ion exchange resin                                                               42    8.3     40.0 15                                                         66    8.3     41.8 99                                                         72    8.3     43.7 234                                                        24    6.3     35.6 9                                 4   After treatment w/ion exchange resin                                                               42    6.3     40.2 18                                                         66    6.3     41.8 157                                                        72    6.3     43.8 306                               __________________________________________________________________________

EXAMPLE IV

This example illustrates the stabilizing effect of sulfite ions on theglucose-isomerizing enzyme under the conditions of an isomerizationreaction.

475 g. of dried filter cake prepared as in example I was suspended insufficient amount of 0.005M cobalt chloride solution to obtain 5 liters.The suspension was adjusted to pH 6.25 and 58° C. and maintained withstirring at these conditions for 6 hours. The suspension was then cooledto room temperature and filtered to obtain a cell-free extract. Thecell-free extract was concentrated tenfold using a Rinco rotaryevaporator. 383 g. of the concentrated cell-free extract was placed in abreaker and the temperature lowered to 1° C., and 255 g. of acetoneadded with stirring to form a precipitate. After 15 minutes, theprecipitate was removed by centrifugation at 2,000 r.p.m. and wasextracted twice with water by suspending in water and centrifuging. Thiswater solution containing the enzyme was dialyzed continuously against10 gallons of demineralized water at 3° C. The dialysate wasconcentrated to 136 g. and was then lyophilized to obtain 15.9 g. ofpurified glucose isomerase preparation having an activity of 14,400GIU/g.

The isomerization mixtures were prepared having the followingcomposition:

3.0M glucose

0.001M cobalt chloride

0.005M magnesium chloride

Sufficient purified glucose isomerase preparation was added to provide11.4 GIU/g. glucose. To one of these isomerization mixtures was addedenough sodium bisulfate to make it 0.005M in respect to this salt (0.096percent SO₂). The mixtures were maintained under nitrogen atmosphere atpH 6.5 and 70° C. Aliquots were removed at the start of theisomerization and after 20, 44 and 92 hours the fructose and colordetermined. The results of these determinations are shown in table V.

Also shown in Table V are residual glucose isomerase activities at thevarious sampling times. The residual enzyme activities in Table V weredetermined as follows:

A test solution was prepared by mixing 25 ml. of isomerate with 25 ml.of a stock solution which was 3M in glucose, 0.2M in pH 6.5 sodiummaleate buffer, 0.02M in magnesium sulfate and 0.001M in cobaltchloride. The test solution was placed in a water-jacketed polarimetercell (20 mm. path). Hot water was circulated through the jacket tomaintain the contents of the cell at 70° C. The cell as placed in aBendix Automatic polarimeter equipped with a recorder and the rate ofchange in optical rotation determined. From the rate of change inoptical rotation, the rate of formation of fructose (V_(f)) catalyzed bythe residual glucose isomerase was calculated. The residual enzymeactivity per gram of dry substance (E/C_(f)) contained in the isomeratewas then calculated according to the following equation:

              V.sub.f (K.sub.s + C - F(1 - K.sub.s /K.sub.p))(C.sub.i +                     C.sub.s)                                                            E/C.sub.i =                                                                             C.sub.g K.sub.f (C - F(1 + 1/K.sub.a))(C.sub.i)                     V.sub.f =rate of fructose formation in moles liter.sup..sup.-1 hr             .sup.-1                                                                       C=total concentration of glucose and fructose in moles per                     liter.                                                                       F=concentration of fructose in moles per liter.                               C.sub.g =concentration of dry substance (g/ml.) in test solution.             k.sub.f =pseudo-first-order rate constant for the breakdown of                 enzyme-glucose complex to enzyme plus fructose (equal                         to 0.012 moles fructose liter.sup..sup.-1 hr.sup..sup.-1 GIU.sup..sup.-1     at pH 6.5 and                                                                  70°C.).                                                               K.sub.s =Michael is constant for substrate 0.580M glucose at pH                6.5 and 70°C.).                                                       K.sub.p =Michaelis constant for product (0.936M fructose at                    pH 6.5 and 70°C.).                                                    K.sub.a =apparent equilibrium constant for the reaction (1.094                 at 70°C.). -C.sub.i =concentration of dry substance (g./ml.) in       isomerate.                                                                    C.sub.s =concentration of dry substance (g./ml.) in stock solu-                tion.                                                                    

Referring to table V it is seen that of the 20 GIU/g. added to theisomerates, 99 and 98 percent were recovered in the 0-hour samples asmeasured by the above technique. At the end of 80 hours the isomeratecontaining the sulfite salt retained 29 percent of the original enzymeactivity whereas the sample containing no sulfite had only 13 percentresidual activity.

                                      TABLE V                                     __________________________________________________________________________                          Enzyme dry                                                     Isomer-        substance                                                                           Residual                                                 ization        ratio in                                                                            activity                                                 time, Percent  isomerate                                                                           (percent of                                       NaHSO.sub.3                                                                          hours fructose                                                                           Color                                                                             (GIU/g.)                                                                            that added)                                       __________________________________________________________________________           0     0.0  7   19.8  99                                                None   20    30.3 27  15.6  78                                                       44    43.4 132 9.8   49                                                       92    48.5 514 2.6   13                                                       0     0.0  3   19.6  98                                                0.005M 20    29.1 5   16.2  81                                                       44    44.3 10  11.1  56                                                       92    50.1 318 5.7   29                                                __________________________________________________________________________

What is claimed is:
 1. A process for enzymatically isomerizing glucosein a glucose-containing liquor to fructose comprising providing aglucose-isomerizing enzyme in a glucose-containing liquor and subjectingthe liquor to isomerizing conditions, there being present in theglucose-containing liquor during isomerization a small amount of awater-soluble salt of sulfurous acid sufficient to measurably reduce theformation of color bodies below that level obtained by carrying out theenzymatic isomerization without the presence of the water-soluble saltof sulfurous acid.
 2. A process for enzymatically isomerizing glucose ina glucose-containing liquor to fructose as defined in claim 1, whereinthe amount of a water-soluble salt of sulfurous acid provided in theglucose-containing liquor during isomerizaton is sufficient to provide alevel of SO₂ in the liquor of from about 0.02 to about 0.3 percent byweight based on the dry substance content of the liquor.
 3. A processfor enzymatically isomerizing glucose in a glucose-containing liquor tofructose as defined in claim 2, wherein the amount of a water-solublesalt of sulfurous acid provided in the glucose-containing liquor duringisomerization is sufficient to provide a level of SO₂ in the liquor offrom about 0.03 to 0.08 percent by weight based on the dry substancecontent of the liquor.
 4. A process for enzymatically isomerizingglucose in a glucose-containing liquor to fructose as defined in claim2, wherein the glucose-isomerizing enzyme is produced from amicro-organism of the Streptomyces genus.
 5. A process for enzymaticallyisomerizing glucose in a glucose-containing liquor to fructose asdefined in claim 4, wherein cellular material containingglucose-isomerizing enzyme is provided in the liquor.
 6. A process forenzymatically isomerizing glucose in a glucose-containing liquor tofructose as defined in claim 5, wherein the pH of the glucose-containingliquor during isomerization is maintained at a value from about 6.5 toabout 7.5.
 7. A process for enzymatically isomerizing glucose in aglucose-containing liquor to fructose as defined in claim 6, wherein thetemperature of the glucose-containing liquor during isomerization ismaintained at a level of from about 50° to about 65° C.
 8. A process forenzymatically isomerizing glucose in a glucose-containing liquor tofructose as defined in claim 7, wherein the glucose-isomerizing enzymeis produced from Streptomyces sp. ATCC 21175 or Streptomyces ATCC 21176.9. A process for enzymatically isomerizing glucose in aglucose-containing liquor to fructose as defined in claim 8, wherein thewater-soluble salt of sulfurous acid provided in the glucose-containingliquor during the isomerization is selected from the group consisting ofmagnesium sulfite, magnesium bisulfite and mixtures thereof. .Iadd. 10.A process for enzymatically isomerizing glucose in a glucose-containingliquor to fructose comprising providing a glucose-isomerizing enzyme ina glucose-containing liquor and subjecting the liquor to isomerizingconditions, there being present in the glucose-containing liquor duringisomerization an amount of a water-soluble salt of sulfurous acidsufficient to reduce denaturation of the glucose-isomerizing enzymebelow that level occurring when the enzymatic isomerizaton is carriedout without the presence of the water-soluble salt of sulfurous acid..Iaddend..Iadd.
 11. A process for enzymatically isomerizing glucose in aglucose-containing liquor to fructose as defined in claim 10, whereinthe glucose-isomerizing enzyme is derived from microorganisms of theStreptomyces genus. .Iaddend..Iadd.
 12. A process for enzymaticallyisomerizing glucose in a glucose-containing liquor to fructose asdefined in claim 11, wherein the microorganisms of the Streptomycesgenus are Streptomyces sp. ATCC
 21175. .Iaddend.